John J.E. Haddad

A non-hypoxic, ROS-sensitive pathway mediates TNF-α-dependent regulation of HIF-1α

A non‐hypoxic, reactive oxygen species (ROS)‐sensitive pathway mediating tumor necrosis factor‐α (TNF‐α)‐dependent regulation of hypoxia‐inducible factor‐1α (HIF‐α) was investigated in vitro. TNF‐α mediated the translocation of HIF‐1α, associated with up‐regulating its activity under normoxia. Analysis of the mode of action of TNF‐α revealed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2 − ) and hydroxyl radical (OH). Antioxidants purported as prototypical scavengers of H2O2 and OH, attenuated TNF‐α‐induced HIF‐1α activation, and blockading NADPH‐oxidase by scavenging O2 − reduced the activity of HIF‐1α. Inhibition of the mitochondrion complex I abrogated TNF‐α‐dependent activation of HIF‐1α. Interrupting the respiratory chain reversed the excitatory effect of TNF‐α on HIF‐1α. These results indicate a non‐hypoxic pathway mediating cytokine‐dependent regulation of HIF‐1α in a ROS‐sensitive mechanism.

Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-α biosynthesis

Redox and ROS regulation of MAPK‐mediated TNF‐α biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS‐mediated TNF‐α secretion is redox‐dependent, NF‐κB‐sensitive and attenuated by N‐acetyl‐L‐cysteine (NAC) and other antioxidants. In alveolar epithelial cells, LPS induced a time‐ and dose‐dependent phosphorylation of MAPKp38. This was associated with the activation of MAPK‐activated protein kinase, which phosphorylated the small heat‐shock protein, Hsp27. MAPKp38 inhibition (SB‐203580) abrogated LPS‐induced TNF‐α production. MAPKERK blockade (PD‐98059) attenuated TNF‐α secretion, an effect synergistically amplified in the presence of SB‐203580. Regulation of NF‐κB by selective inhibitors revealed that this pathway is partially involved in regulating LPS‐mediated TNF‐α secretion. Whereas the proteasome inhibitor, MG‐132, had no effect on LPS‐mediated TNF‐α production, CAPE, sulfasalazine and SN‐50, a cell‐permeant NF‐κB inhibitor, attenuated but did not abrogate TNF‐α biosynthesis. LPS up‐regulated ROS, an effect abrogated by 4′‐hydroxy‐3′‐methoxy‐acetophenone and NAC, which reduced TNF‐α secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPKp38 pathway. ROS induced MAPKp38 phosphorylation and selective antioxidants, including the permeant GSH precursor, γ‐GCE, reduced ROS‐dependent MAPKp38 phosphorylation. These results indicate that the MAPK pathway and MAPK‐mediated regulation of TNF‐α production is redox‐dependent, GSH‐mediated and requires, at least in part, a NF‐κB/ROS‐sensitive mechanism.

Oxygen‐evoked Na+ transport in rat fetal distal lung epithelial cells

1 Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. 2 Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. 3 Reporter gene assays showed that no activation of the α‐ENaC (epithelial Na+ channel α‐subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. 4 Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6‐24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. 5 The increase in GNa thus coincides with activation of the α‐ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.

Alpha-melanocyte-related tripeptide, Lys-d-Pro-Val, ameliorates endotoxin-induced nuclear factor kappaB translocation and activation: evidence for involvement of an interleukin-1beta193-195 receptor antagonism in the alveolar epithelium.

The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB.

Redox regulation of TNF-α biosynthesis: Augmentation by irreversible inhibition of γ-glutamylcysteine synthetase and the involvement of an IκB-α/NF-κB-independent pathway in alveolar epithelial cells

Abstract The pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, contribute to the exacerbation of pathophysiological conditions in the lung. The regulation of cytokine gene transcription involves the reduction–oxidation (redox)-sensitive nuclear factor-κB (NF-κB), the activation of which is mediated through an upstream kinase that regulates the phosphorylation and subsequent degradation of inhibitory-κB (IκB)-α, the major cytosolic inhibitor of NF-κB. It was hypothesised that the lipopolysaccharide (LPS)-induced biosynthesis of TNF-α in vitro is regulated by redox equilibrium. Furthermore, the likely involvement of the IκB-α/NF-κB signalling transduction pathway in regulating LPS-induced TNF-α biosynthesis was unravelled. In a model of alveolar epithelial cells, we investigated the role of l -buthionine-(S,R)-sulfoximine (BSO), a specific and irreversible inhibitor of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in glutathione (GSH) biosynthesis, in regulating LPS-mediated TNF-α production and the IκB-α/NF-κB pathway. Pretreatment with BSO, prior to exposure to LPS augmented, in a dose-dependent manner, LPS-induced TNF-α biosynthesis. In addition, BSO blockaded the phosphorylation of IκB-α, reduced its degradation, thereby allowing its cytosolic accumulation, and subsequently inhibited the activation of NF-κB. These results indicate that there are oxidant-initiated and redox-mediated mechanisms regulating TNF-α biosynthesis and that the IκB-α/NF-κB signal transduction pathway is redox-sensitive but differentially involved in redox-dependent regulation of cytokine signalling in the alveolar epithelium.

The biphasic immunoregulation of pyrimidylpiperazine (Y-40138) is IL-10 sensitive and requires NF-κB targeting in the alveolar epithelium

Pyrimidylpiperazine (Y‐40138), a synthetic derivative of N‐[1‐(4‐{[4‐(pyrimidin‐2‐yl)piperazin‐1‐yl]methyl}phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro‐ and anti‐inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. In alveolar epithelial cells, pre‐treatment (30u2003min) with Y‐40138 reduced LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α, an effect paralleled by up‐regulating an anti‐inflammatory counter‐loop mediated through IL‐10. This differential regulation of pro‐ and anti‐inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF‐κB subunits, particularly NF‐κB1 (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c‐Rel (p75). In addition, Y‐40138 blockaded, in a dose‐dependent manner, the LPS‐induced nuclear activation of NF‐κB. Analysis of the upstream pathway involved in Y‐40138‐dependent retardation of LPS‐induced NF‐κB translocation/activation revealed the involvement of an IκB‐α sensitive pathway. Pre‐treatment with Y‐40138 ameliorated LPS‐induced degradation of IκB‐α in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. Recombinant IL‐10 (0u2003–u200310u2003ngu2003ml−1) blockaded, in a dose‐dependent manner, LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α. Furthermore, rhIL‐10 reduced the DNA binding activity of NF‐κB. Immunoneutralization of endogenous IL‐10 by a polyclonal αIL‐10 (5u2003μgu2003ml−1) reversed the inhibitory effect of Y‐40138 on pro‐inflammatory cytokines and partially restored the DNA binding activity of NF‐κB. These results indicate that Y‐40138 mediated dual immunoregulation of pro‐ and anti‐inflammatory cytokines is IL‐10 sensitive and mediated through the IκB‐α/NF‐κB signal transduction pathway.